Accurate chromosome segregation requires that the spindle attach to a special DNA-protein complex at the centromere called the kinetochore. We have discovered CSE4p, an essential yeast protein required for chromosome segregation that is a homolog of the human centromere protein, CENP-A. Both proteins have a histone fold domain (HFD) like that of core histones. Our goal is to understand CSE4p function in centromere chromatin structure and chromosome segregation. We propose that CSE4p replaces H3 in a subset of specialized nucleosomes which function to organize the centromere DNA into a unique chromatin structure that is necessary for kinetochore assembly. To determine if CSE4p is a nucleosome component we will affinity purify CSE4p protein complexes and identify the components. We will also test whether chromatin containing CSE4p is located at the centromere and how mutations in CSE4 affect chromatin structure and chromosome segregation. Then we will use purified components to assemble functional kinetochores onto reconstituted centromeric chromatin. Understanding centromere function is essential to preventing faulty chromosome transmission in humans, a prominent factor in spontaneous abortions, birth defects and cancer. CSE4p provides us with a unique opportunity to study the special functions of centromeric chromatin in a system where the centromere DNA is well characterized, and where powerful genetic techniques are available. Despite the different kinetochore structures in yeast and mammals, our yeast studies are relevant to understanding chromosome segregation in humans. The HFDs in CSE4p and CENP-A could form nucleosomes at their respective centromeres, leaving the divergent N-termini available to interact with different kinetochore proteins. In this way the chromatin structure underlying the kinetochores would be conserved between species. Clearly, understanding how CSE4p establishes specialized chromatin at the yeast centromere will make a significant contribution to understanding how CENP-A functions in human chromosome segregation.